USPTO Patents Application 10687850

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :
G01N 27/327, 27/333

Al

(11) International Publication Number: WO 98/55853

(43) International Publication Date: 10 December 1998 (10.1Z98)

(21) International Application Number: PCT/AU98/00417

(22) International Filing Date: 3 June 1998 (03.06.98)

(30) Priority Data:

P0 7148^

3 June 1997 (03.06.97)

AU

(71) Applicants (for all designated States except US): AUS-
TRALIAN MEMBRANE AND BIOTECHNOLOGY
RESEARCH INSTITUTE [AU/AU]; 126 Greville Street,
Chatswood, NSW 2067 (AU). THE UNIVERSITY
OF SYDNEY [AU/AU]; Sydney, NSW 2006 (AU).
AUSTRALIAN NATIONAL UNIVERSITY [AU/AU];
Canberra, ACT 2601 (AU). THE GARVAN INSTITUTE
OF MEDICAL RESEARCH [AU/AU]; 384 Victoria Street,
Darlinghurst, NSW 2010 (AU).

(72) Inventors; and

(75) Inventors/Applicants (for US only): ALTIN, Joseph, G.
[AU/AU]; 23 Williamson Street, Holder, ACT 2611
(AU). BURNS, Christopher, John [AU/AU]; 9 North
Street, Balmain, NSW 2041 (AU). PACE, Ronald, John
[AU/AU]; 138 Hawkesbury Crescent, Farrer, ACT 2607
(AU). PARISH, Christopher, R. [AU/AU]; 62 Vassey
Crescent, Campbell, ACT 2612 (AU). FIDDES, Rodney,

John [AU/AU]; 173 Perouse Road, Randwick, NSW 2031
(AU).

(74) Agent: F.B. RICE & CO.; 605 Darling Street, Balmain, NSW
2041 (AU).

(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE,
GH, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ,
PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT,
UA, UG f US, UZ, VN, YU, ZW, ARIPO patent (GH, GM,
KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ,
BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE,
CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC,
NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA,
GN, ML, MR, NE. SN, TD, TG).

Published

With international search report.

(54) Title: RECEPTOR/LIGAND BIOSENSOR

(57) Abstract

The present invention provides a biosensor for use in detecting the oligomerization of receptors. Hie biosensor comprises an electrode
and a bilayer membrane having a top and a bottom layer. The bottom layer is proximal to and connected to the electrode in a manner such
that a space exists between the membrane and the electrode. The membrane comprises a closely packed array of amphiphflic molecules
and a plurality of ion channels comprising first half membrane spanning monomers dispersed in the top layer and second half membrane
spanning monomers dispersed in the bottom layer. The first half membrane spanning monomers are capable of lateral diffusion withm the
upper layer while the second half membrane-spanning monomers are prevented from lateral diffusion within the bottom layer. Receptors
are attached to an end of at least a proportion of the first half membrane-spanning monomers proximal to the surface of the membrane
and the oligomerization of the receptors, causes a change in the conductance or impedance of the membrane. The biosensors are useful in
screening compounds for their ability to promote or mterfere wim receptor oligomerization.

FOR THE PURPOSES OP INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

AL

Albania

ES

Spain

LS

Lesotho

SI

Slovenia

AM

Armenia

FI

Finland

LT

Lithuania

SK

Slovakia

AT

Austria

FR

France

LU

Luxembourg

SN

Senegal

AU

Australia

GA

Gabon

LV

Latvia

SZ

Swaziland

AZ

Azerbaijan

GB

United Kingdom

MC

Monaco

TD

Chad

BA

Bosnia and Herzegovina
Barbados

GB

Georgia

MD

Republic of Moldova

TG

Togo

BB

GH

Ghana

MG

Madagascar

TJ

Tajikistan

BE

Belgium

GN

Guinea

MK

The former Yugoslav

TM

Turkmenistan

BF

Burkina Paso

GR

Greece

Republic of Macedonia

TR

Turkey

BG

Bulgaria

HU

Hungary

ML

Mali

TT

Trinidad and Tobago

BJ

Benin

IE

Ireland

MN

Mongolia

UA

Ukraine

BR

Brazil

IL

Israel

MR

Mauritania

UG

Uganda

BY

Belarus

IS

Iceland

MW

Malawi

US

United States of America

CA

Canada

IT

Italy

MX

Mexico

UZ

Uzbekistan

CF

Central African Republic

JP

Japan

NE

Niger

VN

Viet Nam

CG

Congo

KE

Kenya

NL

Netherlands

YU

Yugoslavia

CH

ownzenana

KG

Kyrgyzstan

NO

Norway

ZW

Zimbabwe

a

Cote d'lvoiro

KP

. Democratic People's

NZ

New Zealand

CM

Cameroon

Republic of Korea

PL

Poland

CN

China

KR

Republic of Korea

PT

Portugal

CU

Cuba

KZ

Kazakstan

RO

Romania

CZ

Czech Republic

LC

Saint Lucia

RU

Russian Federation

DE

Germany

U

Liechtenstein

SD

Sudan
Sweden

DK

Denmark

LK

Sri Lanka

SE

EE

Estonia

LR

Liberia

SG

Singapore

WO 98/55853

PCT/AU98/00417

Receptor/Ligand Biosensor
The present invention relates generally to anchoring molecules to
model membrane systems and to the use of anchored molecules in assays of
inter molecular interactions.

5 Cell surface biomolecules, such as receptors, often need to be in an

oligomerized or clustered form to enable signalling function and interactions
with ligands. Such ligands include growth factors, cytokines, hormones,
surface exposed components on cells, subcellular viral, subviral particle and
other infectious agents (eg Science 275, 1261-64, 1997). By virtue of their

10 ability to undergo multimeric interactions, oligomerized receptors have the
potential to interact stably with ligands of low affinity. Such oligomerization
is usually essential for transmembrane signalling and receptor function.
However, for many receptors the affinity of self-association or interaction
with ligands is not high enough to allow detection using conventional

15 binding techniques, which often require covalent labelling, solubilization
with detergents, or immobilization of the receptor or ligand onto the solid
sensing surface of existing biosensors. These methods are suited to the study
of relatively high affinity interactions and they generally rely on the ability of
the molecules to interact in solution or to maintain stable interaction after

20 cell disruption. Since the effective receptor/ligand concentration in solution
is significantly reduced compared to that on the two-dimensional surface of a
cell (where molecules can oligomerize or cluster and interact stably with
other molecules through multimeric interactions) these methods are limited
in their ability to detect interactions of low affinity.

25 Since oligomerization of receptors is essential for their functional

activity, assay methods which allow detection of this oligomerization process
are highly suitable for screening of compounds that influence receptor
aggregation and hence cellular functions triggered by that process. Such
compounds would be candidates for drugs or other therapeutic agents

30 relevant to disease states in which cellular transmembrane signalling events
are involved. Oligomerization of receptors may be mediated by their
extramembraneous or transmembrane domains.

Atomic force microscopy (also known as scanning probe microscopy)
allows three-dimensional imaging and measurement of structures ranging in

35 size from atomic dimensions to microns, and is revolutionary in its ability to
resolve structures never seen before. The development of optical biosensors

WO 98/55853

PCT/AU98/00417

has permitted the monitoring of the interaction between macromolecules in
real time. To date, both of these techniques generally have been used with
the receptor or the ligand molecule covalently attached to or immobilised
onto a solid surface. Recently a technique has been described in which a
5 linkage of a recombinant hexa-histidine-tagged protein with nitrilotriacetic
acid (NTA) is used to reversibly immobilize a hexa-histidine-tagged protein
onto the solid sensing surface of a BIAcore surface plasmon resonance
biosensor. The formation of a hybrid alkanethiol/phospholipid membrane on
the BIAcore sensing surface also has been described, enabling analysis of the

10 binding of streptavidin to biotinylated phosphatidylethanolamine in the
bilayer. These prior art techniques do not permit an analysis of receptor
interactions under conditions that mimic a cell surface and that allows
lateral mobility of the molecule, as well as the possibility for oligomerization
and multimeric interactions.

15 The present inventors have developed an apparatus which can be

used to detect the oligomerization of receptors, receptor/ligand interaction
and to screen compounds for their ability to interfere or promote such
oligomerization or interaction. The apparatus is best described as a
biosensor in that it involves the use of lipid membranes. Membranes for the

20 use in biosensors have been disclosed In international Patent Application
Nos PCT/AU88/00273, PCT/AU89/00352 PCT/AU90/00025 and
PCT/AU92/00132. The disclosure of these applications is included herein by
reference.

As disclosed in these applications, suitably modified lipid molecules
25 may be caused to assemble into an electrode/ionic reservoir/insulating bilayer
combination that is suitable for incorporation of ion channels and
ionophores. It is also disclosed that the conductance of these membranes is
dependent on the presence or absence of an analyte. In bilayer membranes
in which each layer includes ion channel monomers, the conductance of the
30 membrane is dependent on the lining up of the monomers in each layer to
form continuous ion channels which span the membrane. As these
continuous ion channels are constantly being formed and destroyed, the
conductance of the membrane is dependent on the lifetimes of these
continuous ion channels.
35 In a first aspect the present invention consists in a biosensor for use

in detecting the oligomerization of receptors, the biosensor comprising an

WO 98/55853

PCT/AU98/00417

electrode and a bilayer membrane having a top and a bottom layer, the
bottom layer being proximal to and connected to the electrode in a manner
such that a space exists between the membrane and the electrode, the
membrane comprising a closely packed array of amphiphilic molecules and a
5 plurality of ion channels comprising first half membrane spanning monomers
dispersed in the top layer and second half membrane spanning monomers
dispersed in the bottom layer, the first half membrane spanning monomers
being capable of lateral diffusion within the upper layer and the second half
membrane-spanning monomers being prevented from lateral diffusion within

10 the bottom layer, receptors being attached to an end of at least a proportion
of the first half membrane-spanning monomers proximal to the surface of the
membrane, the oligomerization of the receptors causing a change in the
conductance or impedance of the membrane.

In a preferred embodiment of the present invention the membrane

15 includes membrane spanning amphiphiles which are prevented from lateral
diffusion within the membrane.

In a second aspect, the present invention consists in a biosensor for
use in detecting the oligomerization of receptors, the biosensor comprising
an electrode and a bilayer membrane having a top and a bottom layer, the

20 bottom layer being proximal to and connected to the electrode in a manner
such that a space exists between the membrane and the electrode, the
membrane comprising a closely packed array of amphiphilic molecules,
membrane spanning amphiphiles which are prevented from lateral diffusion
within the membrane and a plurality of ion channels comprising first half

25 membrane spanning monomers dispersed in the top layer and second half
membrane spanning monomers dispersed in the bottom layer, the first half
membrane spanning monomers being capable of lateral diffusion within the
upper layer and the second half membrane-spanning monomers being
prevented from lateral diffusion within the bottom layer, receptors being

30 attached to either an end of at least a proportion of the first half membrane-
spanning monomers proximal the surface of the membrane and to an end of
at least a proportion of the membrane-spanning amphiphiles proximal the
surface of the membrane, the oligomerization of the receptors causing a
change in the conductance or impedance of the membrane.

35 in a third aspect, the present invention consists in a biosensor for use

in detecting receptorAigand interaction, the biosensor comprising an

WO 98/55853

PCT/AU98/00417

electrode and a bilayer membrane having a top and a bottom layer, the
bottom layer being proximal to and connected to the electrode in a manner
such that a space exists between the membrane and the electrode, the
membrane comprising a closely packed array of amphiphilic molecules and a
5 plurality of ion channels comprising first half membrane spanning monomers
dispersed in the top layer and second half membrane spanning monomers
dispersed in the bottom layer, the first half membrane spanning monomers
being capable of lateral diffusion within the upper layer and the second half
membrane-spanning monomers being prevented from lateral diffusion within
10 the bottom layer, receptors being attached to an end of a proportion of the
first half membrane-spanning monomers proximal the surface of the
membrane and ligands being attached to the remainder, the interaction of the
receptors with the ligands causing a change in the conductance or impedance
of the membrane.

15 In a preferred embodiment of the present invention the membrane

includes membrane spanning amphiphiles which are prevented from lateral
diffusion within the membrane.

In a fourth aspect, the present invention consists in a biosensor for
use in detecting receptor/ligand interaction, the biosensor comprising an

20 electrode and a bilayer membrane having a top and a bottom layer, the

bottom layer being proximal to and connected to the electrode in a manner
such that a space exists between the membrane and the electrode, the
membrane comprising a closely packed array of amphiphilic molecules,
membrane spanning amphiphiles which are prevented from lateral diffusion

25 within the membrane and a plurality of ion channels comprising first half
membrane spanning monomers dispersed in the top layer and second half
membrane spanning monomers dispersed in the bottom layer, the first half
membrane spanning monomers being capable of lateral diffusion within the
upper layer and the second half membrane-spanning monomers being

30 prevented from lateral diffusion within the bottom layer, receptors being
attached to either an end of at least a proportion of the first half membrane-
spanning monomers proximal the surface of the membrane or to an end of at
least a proportion of the membrane-spanning amphiphiles proximal the
surface of the membrane, and ligands being attached to the other of at least a

35 proportion of the first half membrane-spanning monomers proximal to the
surface of the membrane or the end of the membrane-spanning amphiphiles

WO 98/55853

PCT/AU98/00417

proximal to the surface of the membrane, receptor/ligand interaction causing
a change in the conductance or impedance of the membrane.

In a fifth aspect the present invention consists in a method of
screening a compound for the ability to interfere with receptor
5 oligomerization or receptor/ligand interaction, the method comprising adding
the compound to the biosensor of the first, second, third or fourth aspect of
the present invention and detecting change in the impedance or conductance
of the membrane.

In a preferred embodiment of the invention, the first and second half
10 membrane spanning monomers are gramicidin or one of its derivatives.

In a further preferred embodiment of the present invention, the
bilayer membrane is attached to the electrode via linking molecules such that
a space exists between the membrane and the electrode. Preferred linking
molecules are those disclosed in application PCT/AU92/00132. In yet a
15 further preferred embodiment of the present invention, the second half
membrane spanning monomers are attached to the electrode via linker
groups.

In yet another preferred embodiment of the present invention, the
bilayer membrane includes membrane spanning lipids, similar to those found

20 in archaebacteria.

Due to ability of the first half membrane spanning monomers to
diffuse laterally within the membrane the biosensors of the present invention
are able to mimic the interactions of receptors as they occur at the cell
surface. The receptors will typically be receptor domains, which may be

25 composed of proteins, protein fragments, proteoglycans, glycoproteins, or
oligosaccharides. The biosensors of the present invention may be also used
to examine interactions between DNA and DNA binding proteins..

Many cellular responses are initiated through the oligomerisation of
30 two or more large protein receptors. Receptors that fall into this categoiy are
the hematopoietic receptor family, epidermal growth factor receptor family,
the insulin receptor family, the colony stimulating receptor family and the
cytokine receptor family. The tethered membrane technology can be used to
screen potential drugs for their ability to block protein-protein interactions,
35 or to cause protein-protein interactions. Specific examples of receptors

WO 98/55853

PCT/AU98/00417

.6

which may be used in the present invention include CD2, CD4, CD48, CD59,

CD94, B7.1, Epo receptors and Erb receptors.

The receptors and ligands may be attached to the membrane using a

variety of strategies a number of which are exemplified in the patent
5 applications referred to above. Other strategies include but are not limited to

metal chelation of a suitably molecularly engineered or chemically attached

terminal group of an expressed protein, glycoprotein, proteoglycan or

oligosaccharide (eg the inclusion of a hexahistidine tag) to a functionalised

moiety on a compound on the membrane surface (eg a metal chelating group
10 such as NTA (nitrilo triacetic acid)). Another possibility is a specifically

engineered receptor or related fragment containing a suitably located peptide

strand which spontaneously inserts into the membrane.

In order that the nature of the present invention may be more clearly

understood preferred forms thereof will now be described with reference to
15 the following non-limiting examples.

EXAMPLES

20 Abbreviations

PBS Phosphate buffered saline

EDC l-(3-Dimethylamino)propyl-3-ethylcarbodiimide hydrochloride

NHS N-hydroxy succinimide

NTA Nitilotriacetic acid

The structure of "linker lipid A" is shown in Figure 1; the structure of
25 "linker gramicidin B" is shown in Figure 2 in which gA is gramicidin, the
structure of which is shown in Figure 3; the structure of MSLOH and
MSL4XB are shown in Figure 4; the structure of "biotinylated gramicidin E"
where n=5, is shown in Figure 5 in which gA is gramicidin, the structure of
which is shown in Figure 3.

30

The cell surface receptor B7.1 (CD80) is a glycoprotein expressed
predominantly on the sin-face of B-cells, dendritic cells, monocytes and
peritoneal macrophages. The receptor is involved in the interaction between
T cells and antigen-presenting cells, and plays an important role in T cell

WO 98/55853

PCT/AU98/00417

7

activation and the induction of antigen-specific immune responses, by
providing a costimulatory signal to the T cells.

The B7.1 receptor has a molecular mass of approximately 60 kDa; it
possesses two extracellular immunoglobulin-like domains, a hydrophobic
5 transmembrane region and a short cytoplasmic tail. The extracellular region
of B7.1 contains binding sites for its cognate receptors CD28 and CTLA-4 on
the surface of T cells. Upon recognition of antigen by the T cell antigen
receptor (TCR), the binding of B7.1 to CD28 provides a co-stimulatory signal
essential for T cell activation. The murine and human B7.1 molecules show

10 considerable phylogenic conservation: human B7.1 can bind and signal

through murine CD28 and CTLA-4, and vice versa. /
The erbB family of growth factor receptors are cell surface receptors
are commonly overexpressed in breast and ovarian carcinomas. The receptor
for EGF, called ErbB-1, can undergo extensive heterodimerisation with three

15 related membrane receptor proteins:- an orphan receptor called ErbB-2, and
two receptors for the Neu differentiation factor ligand (NDF, heregulin, HRG)
called ErbB-3 and ErbB-4. Although no known ligand binds directly to
erbB-2, this receptor appears to be the preferred he terodime rising partner for
the other erbB receptors. The rate of ligand dissociation for a heterodimer

20 containing ErbB-2 is also thought to be slower than for the other dimers.

The experiments described in example 1 and 2 were performed using
a recombinant form of murine B7-1. The recombinant B7.1 used consisted of
the entire amino acid sequence of the extracellular region of murine B7.1 and

25 a hexahistidine (6H) tag at the carboxyl terminal end of the protein

(corresponding to the membrane-proximal end of the native B7.1 protein).
The 6H tag was engineered to enable the receptor to be linked to
nitrilotriacetic acid (NTA) groups through the formation of a metal-chelating
linkage between the six successive histidine residues on the protein and the

30 NTA. The recombinant protein was produced using standard molecular

biology techniques in which the murine B7.1 gene was engineered to encode
the extracellular region of B7.1 with a hexahistidine tag (B7.1-6H); the gene
was then cloned and expressed in insect SF9 and High-5 cells using the
baculovirus expression system. Recombinant B7.1-6H protein was expressed

35 in the culture supernatants of High-5 cells transfected to express the B7.1-6H
gene, and correct expression of the B7.16H protein was confirmed by

WO 98/55853

PCT/AU98/00417

8

SDS-PAGE analysis, and by immunoblotting and immunoprecipitation with
the commercially available murine B7.1-specific monoclonal antibody
16-lOAl. For scale-up expression of the protein, supernatants of High-5 cells
expressing the recombinant B7.1-6H protein were pooled and the
5 recombinant B7.1 protein was purified by Ni-NTA chromatography followed
by gel filtration on FPLC. As judged by SDS-PAGE analysis, the purity of the
B7.1-6H produced by this method was in excess of 95%.

10 Example 1

y

A glass slide was evaporatively coated with a 200A chromium
adhesive layer, followed by a lOOoA layer of gold. The gold coated substrate
was placed in a 50 ml ethanolic solution containing the components as listed
15 in Table 1, in the concentrations shown.

Table 1

COMPONENT

MOLARITY

Linker Lipid A

370 |oM

Mercaptoacetic acid Disulfide

185 jjM

MSL4XB

27.75 nM

MSLOH

5.5 |iM

Linker gramicidin B

55.5nM

20 The gold coated substrate was placed into this solution within five

minutes of preparation. The gold coated substrate was left in this solution
for 60 minutes, and then rinsed copiously with ethanol, and then immersed
in ethanol for 2-3 hours. The gold slide was then rinsed with ethanol and
assembled in an electrode holder such that an electrode is defined, that for

25 the current examples has an area of approximately 11mm 2 . Then, 10 p.1 of an
ethanolic solution of l,2-di(3RS,7R,HR-phytanyl)-glycero-3-phosphocholine
and l,2-di(3RS,7R,llR-phytanyl)glycerol in a 7:3 ratio, 3mM total lipid
concentration, containing biotinylated gramicidin E where n=5, in a
concentration such that the ratio of total lipid to gramicidin derivative is

WO 98/55853

PCT/AU98/00417

9

67,000:1 was added to the surface of the gold electrode and then rinsed with
three washes of 150 |al PBS, leaving 100 jil PBS above the electrode surface.
A silver, counter electrode was immersed in the PBS solution, and the counter
electrode and the sensing electrode connected to an impedance bridge. A DC
5 offset of -300mV was applied to the sensing electrode during AC

measurement. Then 50 jil of 0.1 mg/ml solution of streptavidin bearing
multiple NTA groups (prepared by treating streptavidin with EDC/NHS,
followed by lysine-NTA) was added to the electrode well and left for three to
five minutes, A 5 jil solution of 0.1 mg/ml biotinylated NTA (prepared by

10 reacting lysine-NTA with biotin-NHS) was added and the solution left for a
further 5 minutes, prior to rinsing with PBS (3 x 100 pi). 15 |il of a 0.33
mg/ml solution of the: protein B7, bearing a 6-His tag, was then added and
after 15 minutes, a monoclonal antibody to the B7 protein (5 pi of a 0.7
mg/ml solution) was added. A gating of 8.1% was observed in the impedance

15 spectrum. A control experiment using native streptavidin and omitting the
addition of biotinylated NTA, showed a minimal gating of 2.5%.

Lysine NTA was prepared following the route of Schmidt et al. f
J.Am.Chem.Soc. t 1994, 116, 8485.

20

Example 2

A tethered bilayer membrane was prepared as described in Example 1.

25 The 5 pi of 0.1 mg/ml solution of streptavidin bearing multiple NTA

groups was added to the electrode well and left for three to five minutes. A 5
I-lI solution of 0.1 mg/ml biotinylated NTA (prepared by reacting lysine-NTA
with biotin-NHS) was added and the solution left for a further 5 minutes,
prior to rinsing with PBS (3 x 100 |il). 15 pJ of a 0.33 mg/ml solution of

30 polybiotinylated protein B7 bearing a 6-His tag, (prepared by reacting the 6-
His tagged protein with biotin-NHS) was then added and after 20 minutes, 5
pi. of 0.1 mg/ml solution of streptavidin was added to the electrode well. A
gating of 10.8% was then observed in the impedance spectrum. A control
experiment using native streptavidin and omitting the addition of

35 biotinylated NTA, showed a modest gating of 4.1%.

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10

Example 3

A tethered bilayer membrane was prepared as described in
Example 1. A solution of streptavidin (5|il of 0.1 mg/ml solution) was added
5 to the electrode well and left for three to five minutes and then rinsed with
PBS (3 x 100(il). A solution of biotinylated anti-FLAG Fab' (5^1, 2|iM) was
then added, and after five minutes the well was rinsed with PBS (3 x lOOjal).
A solution of FLAG-labelled HRG (0.2^1 of 0.3^M solution, well
concentration 6nM) was added and the gating of 15% observed by impedance
10 spectroscopy.

./

Example 4

A tethered bilayer membrane was prepared as described in
15 Example 1. A solution of streptavidin (5|il of 0.1 mg/ml solution) was added
to the electrode well and left for three to five minutes and then rinsed with
PBS (3 x lOOp.1). A solution of biotinylated anti-FLAG Fab' (5|il, 2|iM) was
then added, and after five minutes the well was rinsed with PBS (3 x lOO^il).
A 1:1 mixture of FLAG-labelled erbB2 and erbB4 receptors was added to the
20 well (2[il of a 4|uM solution, well concentration 80nM) and after 10 minutes
the well was rinsed with PBS (3 x 100^1). No gating was observed by
impedance spectroscopy at this stage/ A solution of FLAG-labelled HRG
(0.2^1 of 0.3|iM solution, well concentration 6nM) was added and the gating
of —15% was observed by impedance spectroscopy.

25

As will be readily appreciated by those skilled in the art the present
invention provides a method of assaying interactions between membrane
anchored molecules and between anchored molecules and molecules capable
of interacting therewith. More particularly, the present invention is useful to
30 study interactions between receptors and between a receptor and a ligand by
anchoring the extra membranous or transmembrane region of the receptor
biomolecule on to a fluid membrane system.

The present invention thus provides for the anchoring of receptor
molecules onto ion channel containing supported bilayers, that enable the
35 molecules to diffuse laterally and interact This technology is ideal in a
preferred embodiment for studying receptor-receptor and receptor-ligand

WO 98/55853

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11

interactions in a membrane system using ion channels with electrical
impedance detection.

It will be appreciated by persons skilled in the art that numerous
variations and/or modifications may be made to the invention as shown in
5 the specific embodiments without departing from the spirit or scope of the
invention as broadly described. The present embodiments are, therefore, to
be considered in all respects as illustrative and not restrictive.

WO 98/55853 PCT/AU98/00417

12

CLAIMS:-

1. A biosensor for use in detecting the oligomerization of receptors, the
biosensor comprising an electrode and a bilayer membrane having a top and

5 a bottom layer, the bottom layer being proximal to and connected to the
electrode in a manner such that a space exists between the membrane and
the electrode, the membrane comprising a closely packed array of
amphiphilic molecules and a plurality of ion channels comprising first half
membrane spanning monomers dispersed in the top layer and second half

10 membrane spanning monomers dispersed in the bottom layer, the first half
membrane spanning monomers being capable of lateral diffusion within the
xipper layer and the second half membrane-spanning monomers being
prevented from lateral diffusion within the bottom layer, receptors being
attached to an end of at least a proportion of the first half membrane-

15 spanning monomers proximal to the surface of the membrane, the

oligomerization of the receptors causing a change in the conductance or
impedance of the membrane.

2. A biosensor as claimed in claim 1 in which the membrane includes
membrane spanning amphiphiles which are prevented from lateral diffusion

20 within the membrane.

3. A biosensor for use in detecting the oligomerization of receptors, the
biosensor comprising an electrode and a bilayer membrane having a top and
a bottom layer, the bottom layer being proximal to and connected to the
electrode in a manner such that a space exists between the membrane and

25 the electrode, the membrane comprising a closely packed array of
amphiphilic molecules, membrane spanning amphiphiles which are
prevented from lateral diffusion within the membrane and a plurality of ion
channels comprising first half membrane spanning monomers dispersed in
the top layer and second half membrane spanning monomers dispersed in the

30 bottom layer, the first half membrane spanning monomers being capable of
lateral diffusion within the upper layer and the second half membrane-
spanning monomers being prevented from lateral diffusion within the bottom
layer, receptors being attached to either an end of at least a proportion of the
first half membrane-spanning monomers proximal the surface of the

35 membrane and to an end of at least a proportion of the membrane-spanning
amphiphiles proximal the surface of the membrane, the oligomerization of

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PCT/AU98/00417

13

the receptors causing a change in the conductance or impedance of the
membrane.

4. A biosensor for use in detecting receptor/ligand interaction, the
biosensor comprising an electrode and a bilayer membrane having a top and

5 a bottom layer, the bottom layer being proximal to and connected to the
electrode in a manner such that a space exists between the membrane and
the electrode, the membrane comprising a closely packed array of
amphiphilic molecules and a plurality of ion channels comprising first half
membrane spanning monomers dispersed in the top layer and second half

10 membrane spanning monomers dispersed in the bottom layer, the first half
membrane spanning monomers being capable of lateral diffusion within the
upper layer and the second half membrane-spanning monomers being
prevented from lateral diffusion within the bottom layer, receptors being
attached to an end of a proportion of the first half membrane-spanning

15 monomers proximal the surface of the membrane and ligands being attached
to the remainder, the interaction of the receptors with the ligands causing a
change in the conductance or impedance of the membrane.

5. A biosensor as claimed in claim 3 or claim 4 in which the membrane
includes membrane spanning amphiphiles which are prevented from lateral

20 diffusion within the membrane.

6. A biosensor for use in detecting receptor/ligand interaction, the
biosensor comprising an electrode and a bilayer membrane having a top and
a bottom layer, the bottom layer being proximal to and connected to the
electrode in a manner such that a space exists between the membrane and

25 the electrode, the membrane comprising a closely packed array of
amphiphilic molecules, membrane spanning amphiphiles which are
prevented from lateral diffusion within the membrane and a plurality of ion
channels comprising first half membrane spanning monomers dispersed in
the top layer and second half membrane spanning monomers dispersed in the

30 bottom layer, the first half membrane spanning monomers being capable of
lateral diffusion within the upper layer and the second half membrane-
spanning monomers being prevented from lateral diffusion within the bottom
layer, receptors being attached to either an end of at least a proportion of the
first half membrane-spanning monomers proximal the surface of the .

35 membrane or to an end of at least a proportion of the membrane-spanning
amphiphiles proximal the surface of the membrane, and ligands being

WO 98/55853

PCT/AU98/00417

14

attached to the other of at least a proportion of the first half membrane-
spanning monomers proximal to the surface of the membrane or the end of
the membrane-spanning amphiphiles proximal to the surface of the
membrane, receptotfiigand interaction causing a change in the conductance
5 or impedance of the membrane.

7. A biosensor as claimed in any one of claims 1 to 6 in which the first
and second half membrane spanning monomers are gramicidin or one of its
derivatives.

8. A biosensor as claimed in one of claims 1 to 7 in which the bilayer
10 membrane is attached to the electrode via linking molecules such that a

space exists between the membrane and the electrode.

9. A biosensor as claimed in one of claims 1 to 8 in which the second
half membrane spanning monomers are attached to the electrode via linker
groups,

15 10. A method of screening a compound for the ability to interfere with
receptor oligomerization or receptoi/ligand interaction, the method
comprising adding the compound to the biosensor as claimed in any one of
claims 1 to 9 and detecting change in the impedance or conductance of the
membrane.

WO 98/55853

PCT/AU98/00417

1/2

O O

Figure 2

NH

Figure 3

WO 98/55853

PCT/AU98/00417

INTERNATIONAL SEARCH REPORT

International Application No.
PCT/AU 98/00417

CLASSIFICATION OF SUBJECT MATTER

IntCl 6: G01N 27/327, 27/333

According to International Patent Classification (IPC) or to both national classification and IPC

B.

FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)
IPC G01N 27/327, 27/333

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
AU: IPC as above

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
WPAT membran: , recept:

Chem. Abs. (biosensors OR bioelectrodes) AND (membranes) AND (ionophore OR ionophores)

c.

DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

AU 38643/95 A (Australian Membrane & Biotechnology Research Institute)
6 June 1996

AU 59925/96 (692107) (Australian Membrane & Biotechnology Research Institute)
22 January 1997

AU 65327/94 A (Australian Membrane & Biotechnology Research Institute)
8 November 1994

1-10

1-10

1-10

Further documents are listed in the
continuation of Box C

X See patent family annex

* Special categories of cited documents: ^

"A" document defining the general state of the art which is

not considered to be of particular relevance
"E" earlier document but published on or after the "X"

international filing date
"L" document which may throw doubts on priority claim(s)

or which is cited to establish the publication date of "Y"

another citation or other special reason (as specified)
"O" document referring to an oral disclosure, use,

exhibition or other means
T* document published prior to the international filing
date but later than the priority date claimed

later document published after the international filing date or
priority date and not in conflict with the application but cited to
understand the principle or theory underlying the invention
document of particular relevance; the claimed invention cannot
be considered novel or cannot be considered to involve an
inventive step when the document is taken alone
document of particular relevance; the claimed invention cannot
be considered to involve an inventive step when the document is
combined with one or more other such documents, such
combination being obvious to a person skilled in the art
document member of the same patent family

Date of the actual completion of the international search
5 August 1998

Date of mailing of the international search report

- 7 AUG 1998

Name and mailing address of the ISA/AU
AUSTRALIAN PATENT OFFICE
PO BOX 200
WODEN ACT 2606
AUSTRALIA

Facsimile No.: (02) 6285 3929

Authorized officer

ISOBEL TYSON

Telephone No.: (02) 6283 2563

Form PCT/ISA/210 (second sheet) (July 1 992) copgid

INTERNATIONAL SEARCH REPORT

International Application No.
PCT/AU 98/00417

C (Continuation)

DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to
claim No.

AU 56188/94 (663243) (Australian Membrane & Biotechnology Research Institute)
22 June 1994

AU 50334/90 (623747) (Australian Membrane & Biotechnology Research Institute)
24 August 1990

AU 21279/88 (617687) (Commonwealth Scientific & Industrial Research Organisation)
March 1989

Science, vol. 275. 28 February 1997, pp 1261-1264, J. COHEN,
-see Figure on page 1261

1-10

1-10

1-10

1-10

Form PCT/ISA/210 (continuation of second sheet) (July 1992) copgid

A/

INTERNATIONAL SEARCH REPORT

Information on patent family members

This Annex lists the known "A" publication level patent family members relating to the patent documents cited
in the above-mentioned international search report. The Australian Patent Office is in no way liable for these
particulars which are merely given for the purpose of information.

Patent Document Cited in Search Patent Family Member

Report

AU

38643/95

WO

9615454

EP

791176

AU

9500/94

AU

59925/96

WO

9701091

EP

838027

AU

3668/95

AU

65327/94

WO
CA

9424562
2161084

EP

695425

JP

8509807

AU

56188/94

WO
JP

9412875
8504943

EP
US

672251
5591647

CA

2150915

AU

50334/90

WO

9008783

EP

455705

EP

770874

ES

2102364

US

5443955

JP

4504714

DE

69030811

AT

153673

CA

2045640

AU

21279/88

WO

8901159

EP

382736

JP

3503209

JP

2682859

us

5436170

US

5693477

US

5741712

US

5766960

AT

113724

CA

1335879

DE

3852036

END OF ANNEX

International Application No.
PCT/AU 98/00417

Form PCT/lSA/210 (extra sheet) (July 1992) copgid

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